2. Cell membrane Lecture-8: Vesicular traffic (II) Reference: Chapter 14
Lodish Harvey et al. (2008) Molecular Cell Biology (6th edition)
Publisher: W.H. Freeman and Company

3. Exocytosis

4. Constitutive and regulated secretion

5. 25.7 Protein localization depends on further signals
Lysosomes are small bodies, enclosed by membranes, that contain hydrolytic enzymes in eukaryotic cells.

6. 25.7 Protein localization depends on further signals
Figure A transport signal in a trans- membrane cargo protein interacts with an adaptor protein.
A typical cargo protein has a transport signal that is responsible for its entry into budding vesicles. Figure shows that the transport signal in a transmembrane protein is usually a region in its cytoplasmic domain that binds to an adaptor protein of the vesicle coat. Figure shows that the transport signal in a soluble cargo protein (for example, a secreted protein that passes through the lumen) is a region that binds to the lumenal domain of a transmembrane cargo receptor, which in turn has an cytoplasmic domain that binds an adaptor protein. Interaction between the cargo and the coat thus directly or indirectly determines specificity of transport. Such mechanisms control anterograde transport from the ER to the cell surface and other destinations.
蛋白质定位依赖于更深层次的信号
我们认为从内质网通过高尔基体进入或者通过原生质膜进入细胞的进程是任何蛋白质都不可避免的，不会在途中中断：没有信号的情况下，蛋白质通过大批流动连续到达原生质膜。是什么信号对于在每个阶段的识别起作用呢？我们已经有了几种类型的信号的细节：要求一种构像用于蛋白质通过胞吞作用成为细胞内的一部分；一种氨基酸序列使得蛋白质到达内质网；和一种修饰使得蛋白质到达溶酶体（小的膜体，在那里蛋白质降解，见后面）。
通过包被凹陷的受体的内吞要求它们的细胞质内尾部的细节形态信息。NPXY序列（Asn-Pro-X-Tyr）紧靠C端。尽管这是一个基本内吞信号，但是其它细胞质尾部序列影响也是不可忽略的。

7. 25.7 Protein localization depends on further signals
Figure A transport signal in a luminal cargo protein interacts with a transmembrane receptor that interacts with an adaptor protein.
A typical cargo protein has a transport signal that is responsible for its entry into budding vesicles. Figure shows that the transport signal in a transmembrane protein is usually a region in its cytoplasmic domain that binds to an adaptor protein of the vesicle coat. Figure shows that the transport signal in a soluble cargo protein (for example, a secreted protein that passes through the lumen) is a region that binds to the lumenal domain of a transmembrane cargo receptor, which in turn has an cytoplasmic domain that binds an adaptor protein. Interaction between the cargo and the coat thus directly or indirectly determines specificity of transport. Such mechanisms control anterograde transport from the ER to the cell surface and other destinations.

8. Processing to the final form occurs in the secretory vesicle.
Insulin is a good example of a protein that is stored in secretory vesicles until a cell receives an signal to secrete the insulin.
Removal of the Pre-sequence (not shown), folding and disulfide bond formation occur in ER.
Processing to the final form occurs in the secretory vesicle.
This is an example of a protein that you would not want to treat with mercaptoethanol because reduction of disulfide bonds would inactivate the protein.

9. “pre-pro-proteins”
Some proteins are processed in secretory vesicles into multiple small polypeptides. One explanation for this approach is that the small polypeptides are too short to be cotranslationally transported into the ER.

10. 25.7 Protein localization depends on further signals
Figure 25.5 Processing for a complex oligosaccharide occurs in the Golgi and trims the original preformed unit to the inner core consisting of 2 N-acetyl-glucosamine and 3 mannose residues. Then further sugars can be added, in the order in which the transfer enzymes are encountered, to generate a terminal region containing N-acetyl-glucosamine, galactose, and sialic acid.
Enzymes that will be transported to lysosomes are recognized as targets for high mannose glycosylation, and are trimmed in the ER as described in Figure Then mannose-6-phosphate residues are generated by a two-stage process in the Golgi. First the moiety N-acetyl-glucosamine-1-phosphate is added to the 6 position of mannose by GlcNAc-phosphotransferase; then a glucosaminidase removes the N-acetyl-glucosamine (GlcNAc).
注定要运输到溶酶体的酶是在运向内质网的同时被翻译的。它们作为高甘露糖糖基化作用的目标而被识别，在内质网中被修饰，如图34.5中所示。甘露糖-6-磷酸的残基在高尔基体的两步中产生。首先， N-乙酰基氨基葡萄糖-1-磷酸的一半通过GlcNAc-磷酸转移酶被加入到甘露糖的6位置上；然后一个氨基葡糖苷酶移除N-乙酰基葡萄糖胺(GlcNAc)。
酶很大程度上作用于内质网-高尔基体通路（见图34.6）。磷酸转移酶的活动提供了标记溶酶体运输的蛋白质的关键性的一步。这发生在内质网-高尔基体修饰的初期，可能在内质网和顺式高尔基体的之间。酶的特异性的基础在于它识别常见溶酶体蛋白结构的能力，这种结构由基本序列不同的两个短序列组成，但是在三级结构形成一个共同的表面。每一个这种序列都有一个关键性的赖氨酸残余物。这种信号的性质解释了为什么具有很少量“身份”序列的蛋白质共享同一通路来定位。
溶酶体蛋白质继续沿着高尔基体叠层运输直到它们遇到甘露糖-6-磷酸的受体。有两类相关的蛋白质起着受体的作用：一种大的（215kD），一种小的（46kD）。甘露糖-6-磷酸的识别靶向于一个运输包被囊泡到溶酶体的蛋白质。分选到溶酶体的最后阶段发生在高尔基体反式面，在那里由笼型蛋白包被的特殊运输泡收集蛋白质。囊泡运输溶酶体蛋白质到内吞体，在那里它们进入一条通路，运向溶酶体。单个的甘露糖-6-磷酸盐受体库无论它们是新合成的还是被吞噬的可能用于指导蛋白质到达溶酶体，。实际上大多数受体定位在内吞体，在那里它们可以识别被吞噬的蛋白质。
位于内质网内腔的蛋白质在C端有一个短序列，Lys-Asp-Glu-Leu（用词头编码为KDEL）。酵母则使用交替的HDEL或者DDEL信号。如果这个序列被删除了，或者由于其它氨基酸的补充而延伸了，那么这个蛋白质就不再保留在内腔中而是分泌到细胞中了。反过来，如果把这个四肽序列加入到溶菌酶的C端，酶就保持在内质网内腔中，而不是分泌到细胞中去了。这说明有一种能够识别C端四肽以及使它在内腔中定位的机制。

11. Modification of the N-linked oligosaccharides is done by enzymes in the lumen of various Golgi compartments.
1. Sorting in TGN
2. Protection from protease digestion
3. Cell to cell adhesion via selectins

12. 25.8 ER proteins are retrieved from the Golgi
图34.17 同时含有溶酶体和内质网靶向信号的人造蛋白质显示了一条内质网定位的途径。蛋白质被暴露给了第一个而不包括第二个在高尔基体中生成甘露糖－6－磷酸的酶，这之后KDEL序列使得它回到内质网。
Figure An (artificial) protein containing both lysosome and ER-targeting signals reveals a pathway for ER-localization. The protein becomes exposed to the first but not to the second of the enzymes that generates mannose-6-phosphate in the Golgi, after which the KDEL sequence causes it to be returned to the ER.
An interesting question emerges from the behavior of proteins that have an ER-localization signal. Does this signal cause a protein to be held so that it cannot pass beyond the ER or is it the target for a more active localization process? The model shown in Figure suggests that the KDEL sequence causes a protein to be returned to the ER from an early Golgi stack. The same experiment has been performed with KKXX proteins, with similar results.
另一种对于跨膜蛋白质在内质网中的定位有作用的信号是KKXX，它由2个赖氨酸残余物组成，位于细胞质面的尾部，就在C端之前。
蛋白质的行为中有趣的一点是，这些蛋白质具有内质网-定位信号。这个信号是使得蛋白质滞留而不能穿过内质网呢，还是作为另一个更为活跃的定位进程的目标呢？图34.17显示的模型说明KDEL序列导致蛋白质从早期的高尔基体叠层回到内质网。相同的试验已经用带有KKXX的蛋白质做过，得到了类似的结果。
当蛋白质通过高尔基体时，它们的修饰是有序的，因此我们可以使用呈现特殊形式的糖类作为外吐通路上的进程标记。当一个KDEL序列加入到一个蛋白质上，这个蛋白质通常定标在溶酶体（因为它的寡聚糖有了6-甘露糖-P残基），序列使得蛋白质保留在内质网。但是蛋白质由于GlcNac-P仅在高尔基体上补充而被更改。GlcNAc没有移除，所以蛋白质不能进到足够远以穿过高尔基体叠层遇到甘露糖-6-P通路上的第二个酶。这说明KDEL是由一个进入高尔基体后固定的受体所识别的，但是在叠层包含第二个酶之前。
酵母基因ERD1和ERD2的突变阻止了具有HDEL信号的蛋白质在内质网中滞留；相反的，蛋白质被细胞分泌出来。这些基因的产物都是是整合膜蛋白。ERD1的突变引起高尔基体的整体缺陷；这通过从高尔基体中“营救”蛋白质而进行内质网蛋白质分选的过程。ERD2的突变识别了HDEL序列的受体。它的功能模型是它在高尔基体的“营救小室”和内质网中间循环。这一点得到与哺乳动物细胞内受体相一致的定位的证实：在高尔基体有大量发现，但是具有KDEL序列的蛋白质的过分表达使得它在内质网中聚集。这样一个KDEL-蛋白质的结合引起了受体从高尔基体向内质网移动。它对于主要在高尔基体情况下的HDEL序列具有高度的亲和力，但是在内质网情况下具有低的亲和力。这使得ERD2通过它们在高尔基体的HDEL尾部捕获蛋白质，和把它们带回到内质网中，在内质网中释放。
KKXX蛋白质的双赖氨酸残基的模体（motif）是结合在包被体的β’-和α-COP元件上。影响β’、-α、-γ、-COP的酵母的突变体在撤回高尔基体的KKXX蛋白质时是有缺陷的。这说明具有COP-I膜的泡在来自高尔基体的蛋白质的回撤蛋白和使它们回到内质网的过程中是有作用的。

13. 25.8 ER proteins are retrieved from the Golgi
图34.17 同时含有溶酶体和内质网靶向信号的人造蛋白质显示了一条内质网定位的途径。蛋白质被暴露给了第一个而不包括第二个在高尔基体中生成甘露糖－6－磷酸的酶，这之后KDEL序列使得它回到内质网。
Figure An (artificial) protein containing both lysosome and ER-targeting signals reveals a pathway for ER-localization. The protein becomes exposed to the first but not to the second of the enzymes that generates mannose-6-phosphate in the Golgi, after which the KDEL sequence causes it to be returned to the ER.
整体上讲蛋白质的运输不是一个单方向的过程：蛋白质进入内质网和运输穿过高尔基体，除非在途中停顿。正如前面已经提到的，COP-II-包被囊泡提供了从内质网到高尔基体的顺行运输的主要功能。COP-I-包被囊泡提供了沿着高尔基体叠层运输的能力。然而，COP-II-包被囊泡和COP-I-包被囊泡在内质网上的发芽都可观察到，所以每种泡都有运输不同的蛋白质货物的可能。
当用药剂布雷菲德菌素A（BFA）对付细胞时发生逆行运输。药剂阻止了ARF从GDP结合形式修饰为GTP结合形式，结果阻止了包被囊泡的发芽，在高尔基体的囊腔之间形成了微管网络（放弃了它们原先的独立性），并把它们联入到内质网中。进入内质网的顺式间质高尔基体的大多数膜都有再吸收过程，同时伴随着进入内质网中的高尔基体蛋白质的重新分配，实现了逆行运输。
关于这个作用一直很有争议。一种见解是布雷菲德菌素的治疗揭示了一个逆行运输的连续进程，这个过程通常受顺行运输的影响而变得模糊，但是在布雷菲德菌素抑制了顺行运输的时候（因为与顺行运输有关的泡对药物比逆行运输要敏感的多）就显现出来。这说明逆行运输是系统中的一部分，这个系统重新得到膜部件用于补充顺行运输的大批流动，当然也能撤回来自内质网的蛋白质的。
另一种见解是膜表面（包括内质网和高尔基体膜）具有自发的融合能力，但是这通常受到抑制，可能是因为包被体结合在融合位点上。如果包被简单的覆盖着所有的v-SNAREs这是可以做到的，没有能力保护这些地点，融合就可以随意发生，引起了像是人为制造的大范围的逆行运输。当然，正常发生的逆行运输仍然存在，只是规模很小，或者仅仅针对某些特定的蛋白质。
布雷菲德菌素在内质网-高尔基体运输的作用是普遍的，但同时它又抑制了不同细胞中的不同运输进程。在一些细胞类型中，它抑制胞吞转运作用（在极化细胞中从基底外侧表面到顶点运输）；在其它细胞中它抑制从反式高尔基体网络到内吞体的运输。这可能说明有多种的转接子样的蛋白质表征与这些特殊运输进程有关的包被囊泡，只有一些蛋白质可以结合布雷菲德菌素（因此它们的特殊类型泡的功能受到了抑制）。

14. Endocytosed molecules that are destined for the lysosome go from the early endosome to the multivesicular body to the late endosome. Fusion of transport vesicles carrying acid hydrolases from the Golgi causes the late endosome to mature into a lysosome.

15. In some cases, both the receptor and the ligand are transported to the lysosome. This is the case for EGF and its receptor. EGF triggers a cell to proliferate but the signal is only required for a short time. To limit the response time both the receptor and the ligand are removed from the membrane.

16. Mosaic organization of endosomes: subdomains

17. Tubular-vesicular endosomes sort membrane components from lumenal componentsY Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y

18. Experimental demonstration that internalized receptor-ligand complexes dissociate in endosomes
Hepatocyte:
Asialglycoproteins and
their receptor.
Sorting of membrane from
contents: surface area to
volume ratio.
Narrow diameter
tubules

19. Late Endosomes Contain Internal Vesicles
Maturation from early to late endosomes occurs through the formation of multivesicular bodies (MVBs). The MVBs move deeper into the cytoplasm fusing with each other and pre-exisiting late endosomes. These structures are characterized by the formation of internal vesicles.
Vesicles inside of vesicles.

20. Late Endosomes Sort By Selective Internalization of Limiting Membrane
The formation of internal vesicles by pinching off of the limiting membrane of MVBs/late endosomes is a sorting process.
Membrane proteins destined for degradation are marked with a covalent mono-ubiquitin tag.
These mono-ubiquitinated membrane proteins are sorted into invaginating buds that pinch off into internal vesicles.
Internal vesicles and their contents are degraded in the lysosome.

21. The Machinery for MVB formation is used by retroviruses to bud
1. Ubiqutinated Hrs protein on
the endosome recruits Ub-tagged
TM cargo to buds then recruits
ESCRT complexes.
2. ESCRT Required to pinch off
internal vesicles.
3. The Vps4 ATPase disassembles
ESCRT.
4. Ub-Gag mimics Hrs
Recruiting ESCRT to HIV
PM buds.
5. ESCRT pinches off buds
Releasing free virus, and Vps4
Recycles ESCRT.

22. HIV Budding from the cell surface

23. Vesicle budding and fusion
Coated vesicles are formed by polymerization of coat proteins onto a
membrane to form vesicle buds and then pinch off from the membrane
to release a complete vesicle.
Vesicle budding is initiated by recruitment of a GTP-binding proteins:
- ARF protein is for both COPI and clathrin vesicles.
- Sar1 protein is for COPII vesicles.
Vesicles fuse with its target membrane in a process involves interaction
of cognate SNARE proteins.

24. Vesicle budding Step 1: Soluble Sar1-GDP is converted to
Sar1-GTP by Sec12, a GEF on ER membrane.
Binding of GTP causes a conformational
change in Sar1 that exposes its hydrophobic
N-terminus, leading to the anchorage of Sar1
to the ER membrane.
Step 2: Attached Sar1-GTP serves as a
binding site for the Sec23/Sec24 coat protein
complex (COPII subunits). Membrane cargo
proteins are recruited to the vesicle bud by
binding of sorting signal sequence.
Step 3: Once vesicles are released, the
Sec23 subunit promotes Sar1 GTPase
activity and leads to GTP hydrolysis by Sar1.
Step 4: Release of Sar1-GDP from the
vesicle membrane causes disassembly of
the COPII coat.

25. Sorting signals in cargo proteins
For membrane cargo proteins, the vesicle coat selects these proteins
by directly binding to their cytoplasmic sorting signals on cytosolic
portion, while for soluble luminal proteins, the vesicle coat selects these
proteins by indirectly binding to their luminal sorting signals through a
cargo receptor.

26. Regulation of endocytosis
Regulation of endocytosis. Several different kinds of proteins and lipids regulate internalization and endosomal sorting. Rab proteins are membrane associated, Ras-like GTPases that control membrane fusion. Different Rabs are associated with particular endosomes. Inositol phospholipids (phosphoinositides) constitute a small fraction of the phospholipids in the plasma membrane and endosomal membranes. Distinct regions of the plasma membrane and different endosomes are enriched in particular varieties of phosphoinositides which bind with different affinities to proteins with lipid-binding domains. For example, the ENTH domain of Epsin (see below) binds PI(4,5)P2, which is enriched at the plasma membrane in vertebrate cells. Some transmembrane proteins have cytoplasmically located internalization signals that are part of their primary amino acid sequence, and these may bind AP-2. Alternatively, a ubiquitin (Ub) polypeptide that serves as an endocytosis signal may be added posttranslationally to the cytoplasmic domain, and these signals

27. The SNARE complex
During exocytosis of secreted proteins, the v-SNARE is VAMP (vesicle-
associated membrane protein). The t-SNAREs are syntaxin, an integral
membrane protein, and SNAP-25 which is attached to membrane by a
hydrophobic lipid anchor.
The four helices (one from VAMP, one from syntaxin, and two from
SNAP-25) to coil around one another to form a four-helix bundle. The
stability of bundle is hold by the electrostatic interactions of opposite-
charged amino acids between helices.
The dissociation of SNARE complexes requires energy and two proteins,
NSF (NEM-sensitive factor) and α-SNAP (soluble NSF attachment
protein). NSF associates with a SNARE complex with the aid of α-SNAP,
which hydrolyzes ATP and releases energy to dissociate SNARE complex.
t-SNARE
(SNAP-25)
(Syntaxin)
v-SNARE
(VAMP)

28. Vesicles ducking and fusion
Step 1: The ducking between the vesicle and the
target membrane is mediated by the interaction
between the vesicle-attached Rab GTPase and its
effector on the target membrane.
Step 2: VAMP proteins on the vesicle surface
interact with the cytosolic domains of syntaxin
and SNAP-25 on the target membrane to form
a coiled-coil SNARE complex, which brings
two membranes close together.
Step 3: Membrane fusion immediately after the
formation of SNARE complex.
Step 4: NSF associating with α-SNAP binds
to the SNARE complexes. The NSF-catalyzed
hydrolysis of ATP then drives disassembly of
the SNARE complexes. At the same time,
Rab-GTP is hydrolyzed to Rab-GDP and
dissociates from the Rab effector.

29. Vesicle trafficking between ER and cis-Golgi
Step 1-3: the anterograde transport
from the ER to cis-Golgi is mediated
by COPII vesicles. These vesicles
contain newly synthesized proteins
destined for the Golgi, cell surface or
lysosome.
Step 4-6: the retrograde transport
from the cis-Golgi to ER is mediated
by COPI vesicles. The purpose of
this transport is to retrieve v-SNAREs,
membranes and misfolded proteins
back to the ER.

30. KDEL receptor in retrograde transport
Most soluble ER-resident proteins carry
a Lys-Asp-Glu-Leu (KDEL) sequence at
their C-terminus, forming KDEL sorting
signal.
The KDEL sorting signal is recognized
and bound by the KDEL receptor which
is located mainly in the cis-Golgi and in
both COPII and COPI vesicles.
The binding affinity of KDEL receptor is
enhanced at low pH. Thus, the difference
in the pH of the ER and Golgi favors
binding of KDEL-bearing proteins to the
receptor in Golgi-derived vesicles and
their release in the ER.
This retrieval system prevents depletion
of ER luminal proteins such as chaperone
proteins.

31. Models for the polarization of the Golgi
In the vesicular transport model, the Golgi cisternae are static
organelles, which contain their resident proteins. The passing of
molecules from cis to trans through anterograde transport.
In the cisternal maturation model, the Golgi cisternae are dynamic
organelles. Each cisterna matures as it migrates forward. At each
stage, the Golgi-resident proteins carried forward in a cisterna are
moved backward to an earlier compartment by retrograde transport.

32. Tight junctions divide the PM of polarized cells into domains
Apicobasal Polarity is associated with many cell-types.
Epithelial cells form ion-tight monolayers of high electrical resistance.
Apical and Basolateral Domains are different in Lipid and Protein Composition

33. Polarized Cells

34. Membrane trafficking is critical to Polarity
Sorting at the Trans-Golgi
Retention After Secretion
Sorting After Endocytosis
Sorting Signals
Basolateral:
Tyrosine or
DiLeucine
Apical:
N or O-linked
Glycosylation
Or TM domain

35. Three Destinations After Endocytosis In a Polarized Cell

36. Polarized Epithelia Have Apical and Basolateral Specific Endosomes
The additional complexity of the plasma membrane requires extra endosomal compartments for sorting.

37. Basolateral Targeting and Human Disease
Koivisto et al., 2001: In the familial hypercholesterolemia (FH)-Turku LDL receptor allele, a mutation of glycine 823 residue affects the signal required for basolateral targeting in MDCK cells. We show that the mutant receptor is mistargeted to the apical surface in both MDCK and hepatic epithelial cells, resulting in reduced endocytosis of LDL from the basolateral/sinusoidal surface. This work suggests that a defect in polarized LDL receptor expression in hepatocytes underlies the hypercholesterolemia in patients harboring this allele.

38. Processing of N-linked glycosylation in Golgi
The Golgi complex is organized into
3-4 cisternae, which contain different
enzymes for protein glycosylation.
N-linked glycosylation in the Golgi:
> In cis-Golgi, three mannose residues
are removed (1).
> In medial-Golgi, three GlcNAc (2,4)
and one fucose (5) residues are added,
while two mannose (3) residues are
removed.
> In trans-Golgi, three galactose (6)
residues are added, followed by the
linkage of N-Acetylneuraminic acid (7)
on each galactose residue.
Each enzyme move dynamically from
the later to the earlier cisterna through
retrograde vesicle transports.
(GlcNAc)

39. Evidence of Golgi cisternal maturation
 Yeast cells expressing:
> the cis-Golgi protein Vrg4-GFP (green)
> the trans-Golgi protein Sec7-DsRed (red)
A compartment rarely contains both cis- and trans-Golgi
proteins at the same time.

40. Endocytosis Why do cells need endocytosis?
Is there more than one endocytic pathway ?
Clathrin-mediated uptake
Caveolae
Non-clathrin/non-caveolae pathways
Pinocytosis/ Phagocytosis
What are the functional consequences of endocytosis?
How are endocytic structures formed and how do they know where to go?
Where do the textbook models come from?

41. Is cholera toxin internalized to the Golgi complex by a clathrin-dependent process?
Epsin and eps15 mutants inhibit clathrin-mediated transferrin (Tf) uptake to recycling endosomes
Epsin and eps15 mutants do not affect cholera toxin B-subunit (CTXB) uptake to the Golgi complex (marked by b-COP)
Suggests CTXB is delivered to the Golgi complex by a clathrin-independent pathway
b-COP: Marker for the Golgi complex

42. Does internalized CTXB pass through early endosomes?
Early endosome function requires the GTPase Rab5
Dominant negative rab5 S34N (GDP bound) expression perturbs early endosomes and blocks transferrin uptake
Rab5 S34N does not affect delivery of CTXB to the Golgi complex
Suggests CTXB does not pass through early endosomes
Does internalized CTXB pass through early endosomes?
Nichols et al J. Cell Biol.

43. Active Membrane Transport – Review
Process
Energy Source
Example
Active transport of solutes
ATP
Movement of ions across membranes
Exocytosis
Neurotransmitter secretion
Endocytosis
White blood cell phagocytosis
Fluid-phase endocytosis
Absorption by intestinal cells
Receptor-mediated endocytosis
Hormone and cholesterol uptake
Endocytosis via caveoli
Cholesterol regulation
Endocytosis via coatomer vesicles
Intracellular trafficking of molecules

44. The endocytic pathway is divided into the early endosomes and late endosomes pathway.
Materials in the early endosomes are sorted:
Integral membrane proteins are shipped back to the membrane;
Other dissolved materials and bound ligands Multivesicular body (MT mediated transport) the late endosomes.
Dissociation of internalized ligand-receptor complexs in the late endosomes. Molecules that reach the late endosomes are moved to lysosomes.

49. The macromolecules that are degraded in the lysosome arrive by endocytosis, phagocytosis, or autophagy.

50. lysosomes
Lysosomes contain about 40 types of hydrolytic enzymes. For optimal
activity, they need to be activated by proteolytic cleavage and an acidic
environment, which is established by the V-class H+ pumps on lysosomal
membrane.
Mature endosomes containing numerous vesicles in their interior are
usually called multivesicular endosomes. Fusion of a multivesicular
endosome directly with a lysosome releases the internal vesicles into
the lumen of the lysosome, where they can be degraded.
Lysosomal membrane proteins are not incorporated into internal
endosomal vesicles, thus keeping them away from degradation.

51. Formation of multivesicular endosomes
Proteins destined to the multivesicular endosome are tagged with
ubiquitin at the plasma membrane, the TGN or the endosomal membrane.
In the endosomal budding, a ubiquitin-tagged Hrs protein on the
endosomal membrane facilitates loading of ubiquitinated cargo proteins
into vesicle buds and then recruits cytosolic ESCRT proteins to the
membrane (step 1).
The membrane-associated ESCRT proteins act to complete vesicle
budding, leading to
release of a vesicle
carrying cargo into the
endosome (step 2).
ESCRT proteins are
disassembled by the
ATPase Vps4 and
returned to the cytosol
(step 3).

54. Apical-basolateral protein sorting
Proteins destined for either the apical or the basolateral membranes are
sorted in the TGN into different transport vesicles.
When cells are infected with VSV and influenza viruses simultaneously,
the VSV G glycoprotein is found only on the basolateral membrane,
whereas the influenza HA glycoprotein is found only on the apical
membrane.
In hepatocytes, membrane proteins
are directed first to the basolateral
membrane. Then, both apical and
basolateral proteins are
endocytosed in the same vesicles:
> the basolateral proteins are
recycled back to basolateral
membrane.
> the apical proteins are
transported across the
cell to apical membrane
(transcytosis).

55. Intracellular Vesicular Transport

56. Vesicular transport of neurotransmitters

57. Neurotransmission (Latin: transmissio = passage, crossing; from transmitto = send, let through), also called synaptic transmission, is an electrical movement within synapses caused by a propagation of nerve impulses. As each nerve cell receives neurotransmitter from the presynaptic neuron, or terminal button, to the postsynaptic neuron, or dendrite, of the second neuron, it sends it back out to several neurons, and they do the same, thus creating a wave of energy until the pulse has made its way across an organ or specific area of neurons.
Nerve impulses are essential for the propagation of signals. These signals are sent to and from the central nervous system via efferent and afferent neurons in order to coordinate smooth, skeletal and cardiac muscles, bodily secretions and organ functions critical for the long-term survival of multicellular vertebrate organisms such as mammals.
Neurons form networks through which nerve impulses travel. Each neuron receives as many as 15,000 connections from other neurons. Neurons do not touch each other; they have contact points called synapses. A neuron transports its information by way of a nerve impulse. When a nerve impulse arrives at the synapse, it releases neurotransmitters, which influence another cell, either in an inhibitory way or in an excitatory way. The next neuron may be connected to many more neurons, and if the total of excitatory influences is more than the inhibitory influences, it will also "fire", that is, it will create a new action potential at its axon hillock, in this way passing on the information to yet another next neuron, or resulting in an experience or an action.
An example of propagation among neurons is the heart beat. A beat is made when a signal is sent from the Sinoatrial node in a sequence that causes the heart to fully contract emptying all the blood in it and refilling with all new blood. It is important that the pulse is sent out from the SA node because the direction of the pulse between the neurons is what drives the muscle to fully contract. If the pulse comes in from the AV node the heart will stutter and not empty all the blood into the body.

58. Synaptic vesicle and plasma membrane proteins important for vesicle docking and fusion
Lodish et al. Figure 21-31

59. 6. Membrane Potentials and Nerve Impulses
K+ gradients maintained by the Na+-K+ ATPase are responsible for the resting membrane potential.
B. The action potential: The changes in ion channels and membrane potential.
Resting state: All Na+ and K+ channels closed.
Depolarizing phase: Na+ channels open,triggering an action potential.
Repolarizing phase: Na+ channels inactivated, K+ channels open.
Hyperpolarizing phase: K+ channels remain open, Na+ channels inactivated.

60. The sequence of events during synaptic transmission:
Excitable membranes exhibit “all-or-none” behavior.
Propagation of action potentials as an impulse.

61. Cycling of neurotransmitters and synaptic vesicles

62. Cycling of neurotransmitters and synaptic vesicles
The uncoated vesicles employ a variety of antiporters (blue) to import
neurotransmitters (transmitters) from cytosol (step 1).
Transmitter-loaded vesicles move to the active zone (step 2).
Vesicle docks on the membrane of a presynaptic cells, which is mediated
by SNAREs. Synaptotagmin, a Ca+2 sensor for exocytosis of transmitter,
prevents membrane fusion (step 3).
In response to an action potential, voltage-gated Ca+2 channels in
membrane open, allowing an influx of Ca+2 from the synaptic cleft. It
causes a conformational change in synaptotagmin, leading to fusion of
docked vesicles with plasma membrane and release of transmitters into
the synaptic cleft (step 4).
After clathrin/AP vesicles containing v-SNARE and transmitter transporter
proteins bud inward and are pinched off in a dynamin-mediated process,
they loss their coat proteins. At the same time, Na+-transmitter symporters
take up transmitter from the synaptic cleft (step 5).
Vesicles are recovered by endocytosis, creating uncoated vesicles (step
6).

63. 25.6 Budding and fusion reactions
Figure A SNAREpin forms by a 4-helix bundle. Photograph kindly provided by Axel Brunger.
A SNARE complex has a rod-like structure (~4 ⊙ 14 nm) in which the v-SNARE and t-SNARE are bound in parallel. Their membrane anchors are at the same end, implying that the rod must lie in a plane between the two membrane surfaces. This structure is called a SNAREpin. Figure is based on the crystal structure, which shows that the complex consists of a 4-helix bundle. Figure shows a model for the SNAREpin superimposed at the appropriate scale on an electron micrograph of the complex (Weber et al., 1998).
是什么控制着泡的定标的特殊性？当一个囊泡在一个特定的膜上发芽，它有一个特定的目标：离开内质网的泡的目标是顺式高尔基体，离开反式高尔基体的泡与原生质膜融合，等等。用于出芽和融合的器官是无处不在的，所以必须有一些补充的成分使得泡能够识别适当的目标膜。SNARE假说认为识别来源于囊泡携带的s-SNARE膜蛋白与目标膜上的t-SNARE膜蛋白的相互作用。

64. 25.6 Budding and fusion reactions
A SNARE complex has a rod-like structure (~4 ⊙ 14 nm) in which the v-SNARE and t-SNARE are bound in parallel. Their membrane anchors are at the same end, implying that the rod must lie in a plane between the two membrane surfaces. This structure is called a SNAREpin. Figure is based on the crystal structure, which shows that the complex consists of a 4-helix bundle. Figure shows a model for the SNAREpin superimposed at the appropriate scale on an electron micrograph of the complex (Weber et al., 1998).
Figure A SNAREpin complex protrudes parallel to the plane of the membrane. An electron micrograph of the complex is superimposed on the model. Photograph kindly provided by James Rothman.

65. 25.6 Budding and fusion reactions
图34.14 当冲动引起胞吐时，神经递质从供体（突触前膜）细胞释放出来。突触（包被）囊泡与原生质膜相融合， 内容物释放进胞外液。内含物是作用在目标（突触后膜）细胞上的神经递质。
25.6 Budding and fusion reactions
Figure Neurotransmitters are released from a donor (presynaptic) cell when an impulse causes exocytosis. Synaptic (coated) vesicles fuse with the plasma membrane, and release their contents into the extracellular fluid.
Impulses in the nervous system are propagated by the passage of material from a donor (or presynaptic) cell to a recipient (or postsynaptic cell). Figure illustrates a nerve terminal. An impulse in the donor cell triggers the exocytic pathway. Stored coated vesicles (called synaptic vesicles) move to the plasma membrane and release their contents of neurotransmitters into the extracellular fluid. The neurotransmitters in turn act upon receptors at the plasma membrane of the recipient cell.
目标蛋白质的表征需要大量的具有同一目标的泡的分离。突触可以提供这样一个具有这种性质的系统。神经系统受到的刺激通过一条通道从供体（或者突触前）细胞到达受体（或者突触后）细胞。图34.14表示的是一个神经末梢。供体细胞受到的刺激引发了外吐的途径。存储的包被囊泡（叫做突触囊泡）移动到原生质膜上，与它融合，释放它们的神经递质到细胞外液。这些神经递质作用于接受细胞的原生质膜上的受体上。

66. 25.6 Budding and fusion reactions
Figure The kiss and run model proposes that a synaptic vesicle touches the plasma membrane transiently, releases its contents through a pore, and then reforms.
In the "kiss and run" model illustrated in Figure 25.19, a vesicle does not completely fuse with the plasma membrane, but contacts it transiently. The neurotransmitter is released through some sort of pore; then the vesicle reforms. Major questions about this pathway are how the vesicle maintains its integrity and what sort of structure forms the pore.
图34.15扩展了我们关于突触囊泡和突触前细胞的原生质膜显示SNARE蛋白作用的认识。分离的融合微粒在不能水解的ATP类似物存在的条件下可以识别SNARE蛋白。当ATP已经加入时，融合微粒分离，在SNAP上发现的补充成分也释放了。v-SNARE是一种由囊泡携带的跨膜蛋白质。t-SNARE包括两个蛋白质：突触融合蛋白是一种跨膜蛋白质，而SNAP-25由一个脂肪酰基连接在膜上（SNAP-25这个名字有其单独的起源，它与融合微粒的SNAP没有关系）。所有的SNARE都具有暴露在细胞质中的蛋白质的主要部分，细胞质域都具有螺旋－螺旋的结构，这些结构参与蛋白质与蛋白质之间的相互作用。突触的v­­-SNARE直接结合在t-SNAREs上，甚至不需要其它成分的融合微粒。在其它系统中也发现了突触SNAREs的类似物，包括其它动物细胞类型和酵母细胞，我们猜想v-SNARE的识别适当的t-SNARE的能力通常是与对接的专一性有关。

67. 25.6 Budding and fusion reactions
图34.15 结合的特异性是由SNARE细胞提供的。由囊泡携带的v-SNARE识别原生质膜上的t-SNARE. SNARE结合在融合颗粒上。
25.6 Budding and fusion reactions
Figure When synaptic vesicles fuse with the plasma membrane, their components are retrieved by endocytosis of clathrin-coated vesicles.
In the fusion model illustrated in Figure 25.20, the vesicle fuses with the plasma membrane in the conventional manner, releasing its contents into the extracellular space. Recycling occurs by the formation of clathrin-coated vesicles at coated pits, that is, by the endocytic pathway. This may occur at large invaginations of the plasma membrane. The importance of endocytosis in this pathway is emphasized by the fact that inhibition of the formation of the clathrin-coated vesicles affects neurotransmitter release from synaptic vesicles. A major question about the pathway is the relationship between the endocytic and exocytic vesicles. The synaptic vesicles are not clathrin-coated. It is probable that the clathrin-coated endocytic vesicles give rise to synaptic vesicles by losing their clathrin coats, but synaptic vesicles may also form by other pathways (as in the case of AP3-coated vesicles). It is probably true that removal of the clathrin coat takes place quite soon after budding for all classes of clathrin-coated vesicles; the process of removal is not well defined.
v-SNARE与t-SNARE之间的相互作用可能也是调节的一个目标。一种叫做突触结合蛋白的突触蛋白质能够结合在一个3个突触SNARE的复合体上；它的结合是与SNAP的结合是互相排斥的。这样突触结合蛋白能够通过阻止融合微粒的形成来阻止融合过程；它的释放可以引发胞吐作用。

68. 25.6 Budding and fusion reactions
图34.16 Rab 蛋白质影响囊泡运输的特定阶段
25.6 Budding and fusion reactions
Figure Rab proteins affect particular stages of vesicular transport.
Another class of proteins that act only at particular stages of protein transport consists of Rabs. They are attached to membranes via the addition of prenyl or palmityl groups at the C-terminus. There are ~30 Rabs, distributed to different membranous systems in the cell. Figure summarizes their distribution: different Rabs are involved in ER to Golgi transport, in the constitutive and regulated pathways from the Golgi to the plasma membrane, and in stages of transport between endosomes (see also below). For example, mutations in the yeast genes YPT1 or SEC4 that code for two such (related) proteins block transport and cause the accumulation of vesicles in the Golgi stacks or between Golgi and plasma membrane, respectively (for review see Nuoffer and Balch, 1994).
另有一类蛋白质只能在由Rab蛋白组成的蛋白质运输的特殊阶段起作用。它们通过C端的异戊二烯基和棕榈基的加入来附着在膜上。约有30种Rab蛋白，分布在细胞内的各个膜系统中。图34.16总结了它们的分布情况：在从内质网到高尔基体的运输中牵涉到的不同的Rab蛋白，在从高尔基体到原生质膜的构成和调节路径上，和内吞体之间的运输阶段。例如，酵母编码这些相关蛋白的基因YPT1或者SEC4上的突变，会引起运输阻断编码和高尔基体叠层内或是高尔基体和原生质膜之间的囊泡聚集
Rab蛋白是GTP结合蛋白质，在GTP结合的形式下具有活性；但是GTP的水解又使其失去活性。像其他的单价GTP结合蛋白质一样，它们的活性将受到其它影响GTP水解的蛋白质的作用。可能有GAP（GTP水解）对特定的Rab蛋白有活化作用，GEP蛋白质刺激了鸟嘌呤核苷酸的解离，GDI蛋白质阻止了鸟嘌呤核苷酸的离解。
至少一些在特殊运输阶段Rab蛋白质的参与说明Rabs与靶向作用有关，但是它们的功能还不清楚。

69. Endocytosis is a process by which cells take up substances by invaginating the plasma membrane. This process can capture both membrane bound and soluble components.
There are several subclasses of endocytosis:
Phagocytosis takes up large particles and cells.
Pinocytosis continuously takes up small amounts of fluid.
Receptor-mediated endocytosis selectively takes up membrane receptors and associated ligands.
Endocytosis takes up large amounts of the plasma membrane and is balanced by the return of membrane components to the plasma membrane by exocytosis.

72. GenMAPP-generated Ras/ERK signaling pathway shaded to correspond with gene expression data. CAV1=caveolin 1; CAV2=caveolin 2; ER=estrogen receptor

73. Problem based learning

75. Exocytosis: Material (wastes etc
Exocytosis: Material (wastes etc.) are expelled from the cell (recall golgi vesicles).

76. Secretory vesicles concentrate and store products
Secretory vesicles concentrate and store products. Secreted products can be either small molecules or proteins. Proteins originate at the ER. In the Golgi, these proteins aggregate and are packaged into transport vesicles as aggregates.

77. Exocytosis and endocytosis
Exocytosis: a process that a cell releases intracellular molecules
(such as hormones, secretory proteins) contained within a
membrane-bounded vesicle by fusion of the vesicle with its plasma
membrane.
Endocytosis: a process that a cell uptake extracellular material by
engulfing it within cell, including receptor-mediated endocytosis,
phagocytosis and pinocytosis.
Vesicular transport: transport vesicles carrying material as cargo bud
off from the donor compartment and fuse with the target compartment.

78. Vesicular Transport: Exocytosis
Secreting material or replacement of plasma membrane

79. Introduction
图34.2 综述： 膜囊泡从供体小室中出芽，被包被蛋白包围。这些包被蛋白结合到了一个目标小室，解开包被，与目标膜融合，释放出内含物。
Figure 25.2 Vesicles are released when they bud from a donor compartment and are surrounded by coat proteins (left). During fusion, the coated vesicle binds to a target compartment, is uncoated, and fuses with the target membrane, releasing its contents (right).
运输设备由小的膜上的囊泡组成，这些泡的内腔中携带着可溶性蛋白质，囊泡的膜内携带着整合膜蛋白。图34.2解释了出芽和融合的过程的性质，在这些过程中，囊泡在相邻的小室之间移动。每个囊泡从供体表面开始出芽，然后在受体表面融合。它的蛋白质根据性质被释放到内腔或者进入受体的膜中，然后进入新的囊泡中以便转入下一个小室。这一系列的进程都在膜表面间的每个转化中重复着，例如，从内质网膜到高尔基体的转运中，或者高尔基体叠层的嵴之间。
一旦蛋白质进入一个膜性环境，它就嵌在膜内直至到达最终的目的地。进入内质网的膜蛋白以适当的方向插入膜中（对于I组蛋白质，N端在细胞腔内，C端在细胞质内；对于II组蛋白质则方向相反）；蛋白质在系统中移动时一直保持着这个方向。无论蛋白质的目的地是高尔基体、溶酶体还是原生质膜，整个进程都沿同样的路径开始。在每种情况下，蛋白质被运输到包被囊泡中，沿着分泌途径到达适当的目的地，在那里蛋白质的一些结构特征被识别和永久的固定（或被细胞分泌）。
内质网上的蛋白质有两个重要的变化：折叠成适当的构像；通过糖基化被修饰。
蛋白质以未折叠的形式转运到内质网中。折叠是在蛋白质在进入内腔时发生的；可能在蛋白质通过膜时发生一系列结构域的独立折叠。一个50kD的蛋白质的折叠可在3-4分钟内完成，而合成链则需要大约一分钟。
内质网中发生的折叠与蛋白质的修饰有关，并由附属的蛋白质来协助完成。正确的折叠需要加上糖类；实际上这可能是蛋白质修饰的一个重要功能。也许还会有酶PDI（蛋白质二硫键基异构酶）引起的二硫键的修饰。与特殊的内质网蛋白质的连接可能是必须的。一部分或者所有的这些活动都可以在蛋白质进入内质网时由一个酶的复合体来完成，也就是说，必要的功能都与在通过膜的转运位点的蛋白质有关系。折叠和寡聚合作用的自发速度计算说明要使进程足够迅速的在穿过细胞中发生，必须要有辅助的酶的催化作用。
一个具有折叠功能的蛋白质是BiP，是分子伴侣Hsp70 家族的一个成员。BiP协助了寡聚合作用和蛋白质在内质网内腔中的折叠。内质网可能包括几个这样的辅助性蛋白质，它们的功能就是识别蛋白质特殊的折叠形式，和协助蛋白质形成一种构像以转运到下一个目的地。
多价糖蛋白通常在内质网中寡聚合。实际上，寡聚合作用对于进一步的运输是必要的。寡聚物迅速的从内质网被运输到高尔基体，但是不能装配的亚单元或者装配错误的蛋白质则受到阻挡。折叠错误的蛋白质通常与内质网特有的分子伴侣相结合。按照预定的进程，它们会逐渐降解而被移除。这样只有蛋白质折叠正确，在高尔基体或者后续的运输进程中才会发现这个蛋白质。
BiP有两个功能：帮助刚移位的蛋白质产生折叠；移除折叠错误的蛋白质。这些活动能够由同样的反应基本模式产生。假定BiP识别了特定的成熟的，正确折叠的蛋白质构像中的不能接近的多肽序列。这些序列暴露着，在蛋白质以基本的一维模式进入内质网内腔时吸引BiP。如果一个蛋白质折叠错误或者变性了，序列就可能暴露在表面上，而不是象正常情况下被深埋。我们还不清楚一个永久折叠错误的蛋白质是如何以降解作为目标的。

80. Exocytosis Vesicle moves to cell surface Membrane of vesicle fuses
Materials expelled orCell discharges material
Reverse of endocytosis 80

81. Exocytosis (post-Golgi trafficking)
Where do newly synthesized membrane and secretory proteins need to go and how do they get there?
Secretion (constitutive and regulated)
PM protein delivery (polarized and non-polarized cells)
Lysosomal targeting
How are proteins packaged into vesicles, and how do the vesicles know where to go?
What do we know about how the Golgi complex actually works?
Where do the textbook models come from?

82. Exocytosis (post-Golgi trafficking)
Where do newly synthesized membrane and secretory proteins need to go and how do they get there?
Secretion (constitutive and regulated)
PM protein delivery (polarized and non-polarized cells)
Lysosomal targeting
How are proteins packaged into vesicles, and how do the vesicles know where to go?
What do we know about how the Golgi complex actually works?
Where do the textbook models come from?

83. Overview of the secretory/exocytic pathway
Recycling vesicles
Transitional ER site
COPI vesicles
To plasma membrane
To secretory granules
COPII vesicles
cis
medial
trans
To endosomes
TGN
TGN = trans-Golgi network

84. Regulated secretion Occurs in endocrine, exocrine and neuronal cells
Insulin secretion in pancreatic b-cells
Trypsinogen secretion in pancreatic acinar cells
Exocytosis occurs in response to a trigger (ex. Ca2+)
5-48

85. Processing of regulated secretory proteins
Proteins undergo proteolytic processing from a proprotein to the mature form
Processing occurs in secretory vesicles as they move away from the TGN
Undergo selective aggregation with one another that aids in their sorting
Proteins become highly concentrated (condensed)  dense-core granules

86. Insulin in the regulated secretory pathway
Antibody binds proinsulin (not insulin)
Antibody binds insulin (not proinsulin)
Mature secretory vesicles
Mature secretory vesicles
Clathrin coat
Golgi complex
Golgi complex
Immature secretory vesicles
Immature secretory vesicles
Vesicle budding from TGN
Vesicle budding from TGN
17-41

87. Constitutive secretion/ exocytosis of plasma membrane proteins
Delivered via membrane vesicles directly from the TGN to the cell surface
Share same vesicles as constitutively secreted proteins
Remarkably little is known about how plasma membrane proteins are sorted into secretory vesicles
May be more than one class of carrier vesicles
VSVGts045, a model protein for studying the secretory pathway (shown here tagged with GFP)

88. Visualizing the secretory pathway: Fusion of TGN-derived vesicles containing VSVGts045-CFP or YFP-GL-GPI with the plasma membrane observed in living cells using total internal reflection microscopy Keller et al (2001) Nature Cell Biol.

89. Exocytosis in polarized epithelial cells
The functions and thus protein composition of the apical and basolateral domain differ
Proteins and lipids must be delivered to the correct PM domain
Proteins can be sorted directly from the TGN to the apical or basolateral domain
Proteins can also be delivered indirectly by transcytosis
Mostov et al Cell 99:

90. “ Direct” versus “indirect” (transcytotic) trafficking in polarized cells
Lodish et al. Figure 17-43

92. Transcytosis
In the infant intestine, antibodies are ingested from mother’s milk.
They bind to Fc receptors on the apical surface of the intestine.
The IgG-FcR complex is transcytosed to the basolateral side where the IgG is released.
The empty FcR is then transcytosed
back to the apical side.
The pH values on either side of the
epithelium are critical for correct binding and release.

93. Transcytosis provides a way to deliver proteins across an epithelium.
Transport of antibodies in milk across the gut epithelium of baby rats.
Acidic pH of the gut favor association of antibody with Fc receptor whereas the neutral pH of the extracellular fluid favors dissociation.

94. Transcytosis: a closer look
lumen
Transcytosis: transport of macromolecular cargo from one side of the cell to the other
Transcytosis is also utilized in the biosynthetic trafficking of some PM proteins
pIgA-receptor is a model for studying transcytosis
Contains sorting information in its cytoplasmic tail
pIgA is secreted into the the gut lumen, bile and milk as part of the mucosal immune response
pIgA-R
pIgA
Blood/interstitial
synthesizes IgA
Tuma and Hubbard (2003)

95. Ras trafficking via the secretory pathway
Ras is targeted to the plasma membrane by its C-terminal domain
CAAX
Second signal (polybasic or palmitoylation)
The CAAX motif targets the protein to the ER
PM delivery of HRas but not KRas is blocked by BFA
Indicates HRas relies on vesicular transport to reach the cell surface
HRas
KRas
Magee and Marshall (1999) Cell

96. Blocks to secretion/ PM protein delivery
20º C (mechanism unknown, but it works!)
Brefeldin A (inhibits assembly of COPI vesicles; blocks ER-to-Golgi trafficking)
Cholesterol depletion (disrupts lipid rafts)
Sec mutants (yeast)
Microinjection of antibodies against regulatory proteins

97. 25.7 Protein localization depends on further signals
Lysosomes are small bodies, enclosed by membranes, that contain hydrolytic enzymes in eukaryotic cells. 97

98. Lysosomal trafficking
Trafficking of soluble lysosomal hydrolases
Hydrolases are modified by mannose-6-phosphate (M6P) in the cis-Golgi
The M6P receptor captures the hydrolases in the TGN as the receptor cycles between the TGN and late endosomes in clathrin-coated vesicles (AP-1, GGA)
The phosphate is removed from hydrolases in late endosomes to prevent recycling of the hydrolases with the M6P receptor
Secreted hydrolases are captured and delivered to lysosomes by endocytosis via PM-localized M6P receptors
Trafficking of lysosomal membrane proteins
Sorting information is contained in their cytoplasmic tails

99. The Lysosome
The endpoint of the endocytosis pathway for many molecules is the lysosome, a highly acidic organelle rich in degradative enzymes.
The V-ATPase maintains the high acidity of the lumen by pumping protons across the lipid bilayer.

100. Trafficking of lysosomal hydrolases to lysosomes by the mannose-6-phosphate receptor

101. Exocytosis (post-Golgi trafficking)
Where do newly synthesized membrane and secretory proteins need to go and how do they get there?
Secretion (constitutive and regulated)
PM protein delivery (polarized and non-polarized cells)
Lysosomal targeting
How are proteins packaged into vesicles, and how do the vesicles know where to go?
What do we know about how the Golgi complex actually works?
Where do the textbook models come from?

102. Key steps in the formation of clathrin-coated vesicles
Activation (TGN)
Activation (PM)
Cargo capture
Coat assembly
Scission
Uncoating
Kirchhausen 2000 Nature Reviews Molecular Cell Biology 1:187

103. Making and moving vesicles: general sorting and trafficking machinery
Cargo sorting signals
Membrane lipids
Vesicle formation- clathrin and accessory proteins
Cargo capture- adaptors
“Pinchase”- dynamin
Direct vesicle movement- actin, microtubules and motors
Vesicle targeting and fusion machinery- Rabs, SNARES
Docking sites on the plasma membrane in polarized cells- exocyst
Lodish et al. Figure 17-51

104. Sorting signals in cargo molecules
Signal sequence
Type of protein
Transport step
Vesicle type
Signal receptor
Mannose-6-phosphate
Secreted
(lysosomal)
TGN to PM and late endosome
clathrin
M6P-R, AP1 and AP2
Tyr-X-X-Ø
membrane
(endosome, BL)
PM to endosome
AP2, AP1B
Leu-Leu (LL)
Selective aggregation
secreted (regulated)
TGN to secretory granule ?
GPI-anchor
(apical)
TGN to PM
unknown
Lipid rafts/?

105. Exocytosis (post-Golgi trafficking)
Where do newly synthesized membrane and secretory proteins need to go and how do they get there?
Secretion (constitutive and regulated)
PM protein delivery (polarized and non-polarized cells)
Lysosomal targeting
How are proteins packaged into vesicles, and how do the vesicles know where to go?
What do we know about how the Golgi complex actually works?
Where do the textbook models come from?

106. The Golgi complex Central organelle of the secretory pathway
Comprises stacks of flattened cisternae
Contains resident enzymes that modify newly synthesized proteins and lipids (ex. glycosylation)
At the trans most stack, proteins are sorted for delivery inside the cell or for secretion
Golgi morphology and composition is maintained despite the flux of proteins and lipids in the secretory pathway
The Golgi complex
3D EM tomography of the Golgi complex

107. Nothing is simple when it comes to the Golgi complex
How does cargo move through the Golgi complex?
Cisternal maturation vs vesicular transport
How is the Golgi complex inherited during mitosis?
ER absorption vs vesiculation
How does the Golgi complex form?
Self-organizes following ER export vs. stable matrix which nucleates formation

108. Models for transport through the Golgi
Vesicular transport
Cisternal maturation
Interlinked network
Elsner et al 2003

109. What is the fate of the Golgi in mitosis?
Barr, 2004

110. Exocytosis (post-Golgi trafficking)
Where do newly synthesized membrane and secretory proteins need to go and how do they get there?
Secretion (constitutive and regulated)
PM protein delivery (polarized and non-polarized cells)
Lysosomal targeting
How are proteins packaged into vesicles, and how do the vesicles know where to go?
What do we know about how the Golgi complex actually works?
Where do the textbook models come from?

111. 25.10 Summary
1. Proteins that reside within the reticuloendothelial system or that are secreted from the plasma membrane enter the ER by cotranslational transfer directly from the ribosome. 2. Proteins are transported between membranous surfaces as cargoes in membrane-bound coated vesicles. 3. Modification of proteins by addition of a preformed oligosaccharide starts in the endoplasmic reticulum. 4. Different types of vesicles are responsible for transport to and from different membrane systems. 5. COP-I-coated vesicles are responsible for retrograde transport from the Golgi to the ER. 6. COP-II vesicles undertake forward movement from the ER to Golgi. 总结
蛋白质以顺行方向从内质网穿过高尔基体传输。除非它们拥有了特定的内质网保留信号，则进入内质网的蛋白质继续大量流动流向高尔基。逆向传输没有很好的表征，但是残留在内质网中的蛋白质是利用特定的信号从它们的高尔基体中撤回的。C-端 KDEI就是一个很好的例子。从内质网到高尔基体或原生质膜没有膜的净流动。所以随着大量流动的膜移动必须返回到内质网中。
增加其它预制的寡聚糖的蛋白质修饰开始在内质网。高甘露寡聚糖被修饰。复杂的寡聚糖通过进一步修饰而产生，这些改变是在通过高尔基体传输时发生的，由蛋白质碰到的酶在高尔基体叠层上的定位而定。蛋白质在反式高尔基体中依据不同的目的地被分选。溶酶体的分选信号就是甘露糖-6-磷酸的存在。
蛋白质从内质网起，沿着高尔基体叠层，像在膜结合包被囊泡中的货物一样被运输。这个囊泡形成于供体膜的出芽阶段：它们和目标膜融合时卸下它们的货物。当囊泡形成时，加上了蛋白质包被，而且必须在它们与目标膜融合前被移除。在内质网-高尔基体系统中发现了两种类型的囊泡，分别为COP-I和COP-II膜。COP-I包被囊泡被外被体所包围。一种外被体蛋白质B-COP是与笼型蛋白包被囊泡的B-adaptin相关的，说明这是在COP-I-膜和笼型蛋白包被的囊泡之间的一种共同结构的可能性。COP-II-包被囊泡涉及在顺行传输中；而COP-I-膜囊泡涉及在逆行传输中。

112. 25.10 Summary
7. In the pathway for regulated secretion of proteins, proteins are sorted into clathrin-coated vesicles at the Golgi trans face. 8. Budding and fusion of all types of vesicles is controlled by a small GTP-binding protein. 9. Vesicles recognize appropriate target membranes because a vSNARE on the vesicle pairs specifically with a tSNARE on the target membrane Receptors may be internalized either continuously or as the result of binding to an extracellular ligand The acid environment of the endosome causes some receptors to release their ligands; the ligand are carried to lysosomes, where they are degraded, and the receptors are recycled back to the plasma membrane by means of coated vesicles.
在受调节的蛋白质分泌的途径上，蛋白质在反式高尔基体面上，被分选进入笼型蛋白包被囊泡中。一些囊泡可以融合成为分泌微粒。囊泡也可以移动到内吞体中可以控制运输到细胞表面。分泌的囊泡可以通过细胞外信号被激发在原生质膜处卸下它们的货物。相似的囊泡可以用于胞吞，通过这种途径蛋白质可以从细胞表面被内含化。在笼型蛋白包被囊泡中，内部的包被包含结合素蛋白，它是结合在笼型蛋白上的：在内吞囊泡中发现了B-结合素。B-结合素使得从高尔基移动到内吞体的囊泡具有一定的特征。
受体可能被持续内含化，或者作为一个结合在细胞外的配基而内含化。当受体侧向移动到包被小窝时，受体介导的胞吞作用开始。受体的胞浆结构域有一个蛋白质识别被认为是与包被小窝有关的信号。一个暴露的定位在跨膜域附近的酪氨酸是一个共同的信号：它可以是序列NPXY的一部分信号。当受体进入包被小窝时，笼型蛋白-包被夹断形成囊泡，然后它将移入早期内吞体。
内吞体的酸性环境引起了一些受体释放它们的配基：这些配基被传输到溶酶体，在那里它们被降解。受体以包被囊泡的方式循环回到原生质膜。一个没有分离的配基可以和它的受体再循环。在某些情况下，受体-配基复合物被传输到溶酶体，并被降解。

113. A simple experiment shows that many sorting signals consist of a continuous stretch of amino acid sequence called a “signal sequence”
Fusing sorting signals to GFP is particularly good way to do this experiment.

114. GFP

115. Cytoplasmic Nuclear

116. Actin-GFP
PAX-GFP